Inhibitors of c-Met protein kinase

ABSTRACT

The present invention relates to compounds of formula I, which is useful in the inhibition of c-Met protein kinase. The invention also provides pharmaceutically acceptable compositions comprising compounds of formula I and methods of using the compositions in the treatment of proliferative disorders.

This present application claims the benefit, under 35 U.S.C. §119, toU.S. Provisional Application No. 61/181,778, filed May 28, 2009 theentire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to selective inhibitors of c-Met. Theinvention also provides pharmaceutically acceptable compositionscomprising a c-Met inhibitor and methods of using the compositions inthe treatment of various proliferative disorders.

BACKGROUND OF THE INVENTION

Hepatocyte growth factor (HGF), also known as scatter factor, is amulti-functional growth factor that enhances transformation and tumordevelopment by inducing mitogenesis and cell motility. Further, HGFpromotes metastasis by stimulating cell motility and invasion throughvarious signaling pathways. In order to produce cellular effects, HGFmust bind to its receptor, c-Met, a receptor tyrosine kinase. c-Met, awidely expressed heterodimeric protein comprising of a 50 kilodalton(kDa) α-subunit and a 145 kDa alpha-subunit (Maggiora et al., J. CellPhysiol., 173:183-186, 1997), is overexpressed in a significantpercentage of human cancers and is amplified during the transitionbetween primary tumors and metastasis. The various cancers in whichc-Met overexpression is implicated include, but are not limited to,gastric adenocarcinoma, renal cancer, small cell lung carcinoma,colorectal cancer, prostate cancer, brain cancer, liver cancer,pancreatic cancer, and breast cancer. c-Met is also implicated inatherosclerosis and lung fibrosis.

Accordingly, there is a great need to develop compounds useful asinhibitors of c-Met protein kinase receptor. In particular, preferredcompounds should have high affinity to the c-Met receptor and showfunctional activity as antagonists, while showing little affinity forother kinase receptors or for targets known to be associated withadverse effects.

SUMMARY OF THE INVENTION

It has been found that6-((R)-1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)ethyl)quinolineand5,7-difluoro-6-((R)-1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)ethyl)quinolineare effective in the inhibition of c-Met and are surprisingly lessactive against PDE3 than their respective enantiomers.

Accordingly, the invention features a compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen orfluoro.

The invention also provides pharmaceutical compositions that include acompound of formula I and a pharmaceutically acceptable carrier,adjuvant, or vehicle. In addition, the invention provides methods oftreating or lessening the severity of a proliferative disease,condition, or disorder in a patient that includes the step ofadministering to the patient a therapeutically effective dose of acompound of formula I, or a pharmaceutical composition thereof.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Terminology

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, and the Handbook of Chemistry and Physics, 75^(th) Ed. 1994.Additionally, general principles of organic chemistry are described in“Organic Chemistry,” Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

Description of the Compound of the Invention

In a first aspect, the invention features the following compounds offormula I:

or pharmaceutically acceptable salts thereof, wherein each R¹ is,independently, hydrogen or fluoro.

In one embodiment R¹ is hydrogen. In another embodiment, R¹ is fluoro.

In another aspect, the invention features a pharmaceutical compositioncomprising a compound of formula I, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier, adjuvant orvehicle. In one embodiment, the composition includes an additionalchemotherapeutic or anti-proliferative agent, an anti-inflammatoryagent, an agent for treating atherosclerosis, or an agent for treatinglung fibrosis.

In another aspect, the invention features a method of treating orlessening the severity of a proliferative disorder in a patientcomprising administering a compound of formula I in an amount sufficientto treat or lessen the severity of a proliferative disorder in saidpatient. In one embodiment, the proliferative disorder is metastaticcancer. In another embodiment, the proliferative disorder is aglioblastoma; hepatocellular carcinoma, a gastric carcinoma; or a cancerselected from colon, breast, prostate, brain, liver, pancreatic or lungcancer.

Compositions, Formulations, and Administration of Compounds of theInvention

In another aspect, the invention provides a composition comprising acompound of formula I or a pharmaceutically acceptable derivativethereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle.In one embodiment, the amount of compound in a composition of thisinvention is such that is effective to measurably inhibit c-Met in abiological sample or in a patient. Preferably the composition of thisinvention is formulated for administration to a patient in need of suchcomposition. Most preferably, the composition of this invention isformulated for oral administration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

It will also be appreciated that the compounds of formula I can exist infree form for treatment, or where appropriate, as a pharmaceuticallyacceptable derivative thereof. According to the present invention, apharmaceutically acceptable derivative includes, but is not limited to,pharmaceutically acceptable prodrugs, salts, esters, salts of suchesters, or any other adduct or derivative which upon administration to apatient in need is capable of providing, directly or indirectly, acompound of formula I as otherwise described herein, or a metabolite orresidue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 66:1-19, 1977, which isincorporated herein by reference. Pharmaceutically acceptable salts ofcompounds of formula I include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. In Remington: TheScience and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, are disclosed various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with a compound of formulaI, such as by producing any undesirable biological effect or otherwiseinteracting in a deleterious manner with any other component(s) of thepharmaceutically acceptable composition, its use is contemplated to bewithin the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal, intraocular,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of compounds of formula I include, but are not limitedto, mineral oil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutically acceptable compositions can beformulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated, e.g., as micronized suspensions in isotonic, pH adjustedsterile saline or other aqueous solution, or, preferably, as solutionsin isotonic, pH adjusted sterile saline or other aqueous solution,either with or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutically acceptablecompositions may be formulated in an ointment such as petrolatum. Thepharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of formula I, it is oftendesirable to slow the absorption of this compound from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of a compound of formula I thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, dissolving orsuspending a compound of formula I in an oil vehicle accomplishesdelayed absorption of a parenterally administered compound form.Injectable depot forms are made by forming microencapsule matrices of acompound of formula I in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of compound topolymer and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping a compound offormula I in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing a compound of formula Iwith suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound offormula I include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants or patches. The active component is admixedunder sterile conditions with a pharmaceutically acceptable carrier andany needed preservatives or buffers as may be required. Ophthalmicformulation, eardrops, and eye drops are also contemplated as beingwithin the scope of this invention. Additionally, the present inventioncontemplates the use of transdermal patches, which have the addedadvantage of providing controlled delivery of a compound of formula I tothe body. Such dosage forms can be made by dissolving or dispensing acompound of formula I in the proper medium. Absorption enhancers canalso be used to increase the flux of a compound of formula I across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing a compound of formula I in a polymer matrix orgel.

Compounds of formula I are preferably formulated in dosage unit form forease of administration and uniformity of dosage. The expression “dosageunit form” as used herein refers to a physically discrete unit of agentappropriate for the patient to be treated. It will be understood,however, that the total daily usage of a compound of formula I andcompositions comprising a compound of formula I will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts.

The amount of a compound of formula I that may be combined with thecarrier materials to produce a composition in a single dosage form willvary depending upon the host treated, the particular mode ofadministration. Preferably, the compositions should be formulated sothat a dosage of from 0.01 to 100 mg/kg body weight/day of the inhibitorcan be administered to a patient receiving these compositions. In oneexample, compositions are formulated such that the dosage of a compoundof formula I is from 5 to 30 mg/kg body weight/day.

Depending upon the particular condition, or disease, to be treated orprevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may also be present inthe compositions of this invention. As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated.” Examples of additionaltherapeutic agents are provided infra.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Uses of Compounds of Formula I and Compositions Comprising Compounds ofFormula I

According to one embodiment, the invention relates to a method ofinhibiting c-Met protein kinase activity in a biological samplecomprising the step of contacting said biological sample with a compoundof formula I, or a composition comprising said compound. The term“biological sample,” as used herein, means a sample outside a livingorganism and includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereofand blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. Inhibition of kinase activity in a biological sampleis useful for a variety of purposes known to one of skill in the art.Examples of such purposes include, but are not limited to, biologicalspecimen storage and biological assays. In one embodiment, the method ofinhibiting kinase activity in a biological sample is limited tonon-therapeutic methods.

The term “c-Met” is synonymous with “c-MET,” “cMet”, “MET”, “Met” orother designations known to one skilled in the art.

According to another embodiment, the invention relates to a method ofinhibiting c-Met kinase activity in a patient comprising the step ofadministering to said patient a compound of formula I, or a compositioncomprising said compound.

The term “c-Met-mediated disease” or “c-Met-mediated condition”, as usedherein, means any disease state or other deleterious condition in whichc-Met is known to play a role. The terms “c-Met-mediated disease” or“c-Met-mediated condition” also mean those diseases or conditions thatare alleviated by treatment with a c-Met inhibitor. Such conditionsinclude, without limitation, renal, gastric, colon, brain, breast,prostate, liver, pancreatic, or lung cancer, glioblastoma,atherosclerosis, or lung fibrosis.

In one aspect, the present invention features a method treating aproliferative disorder in a patient comprising the step of administeringto the patient a therapeutically effective dose of a compound of formulaI or a composition comprising a compound of formula I.

According to one embodiment, the proliferative disorder is cancer, suchas, for example, renal, gastric, colon, brain, breast, liver, prostate,and lung cancer, or a glioblastoma.

In another embodiment, the present invention relates to a method oftreating or lessening the severity of hepatocellular carcinoma in apatient in need thereof, comprising administering to said patient acompound of formula I or composition thereof.

In another embodiment, the proliferative disorder is polycythemia vera,essential thrombocythemia, chronic idiopathic myelofibrosis, myeloidmetaplasia with myelofibrosis, chronic myeloid leukemia (CML), chronicmyelomonocytic leukemia, chronic eosinophilic leukemia,hypereosinophilic syndrome, systematic mast cell disease, atypical CML,or juvenile myelomonocytic leukemia.

In another embodiment, the proliferative disorder is atherosclerosis orlung fibrosis.

Another aspect of the present invention relates to a method ofinhibiting tumor metastasis in a patient in need thereof, comprisingadministering to said patient a compound of formula I or a compositionthereof.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents that are normally administered to treatthat condition may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated”.

In one embodiment, chemotherapeutic agents or other anti-proliferativeagents may be combined with a compound of formula I to treatproliferative diseases and cancer. Examples of known chemotherapeuticagents include, but are not limited to, alkylating agents, such as, forexample, cyclophosphamide, lomustine, busulfan procarbazine, ifosfamide,altretamine, melphalan, estramustine phosphate, hexamethylmelamine,mechlorethamine, thiotepa, streptozocin, chlorambucil, temozolomide,dacarbazine, semustine, or carmustine; platinum agents, such as, forexample, cisplatin, carboplatinum, oxaliplatin, ZD-0473 (AnorMED),spiroplatinum, lobaplatin (Aeterna), carboxyphthalatoplatinum,satraplatin (Johnson Matthey), tetraplatin BBR-3464, (Hoffmann-LaRoche), ormiplatin, SM-11355 (Sumitomo), iproplatin, or AP-5280(Access); antimetabolites, such as, for example, azacytidine, tomudex,gemcitabine, trimetrexate, capecitabine, deoxycoformycin,5-fluorouracil, fludarabine, floxuridine, pentostatin,2-chlorodeoxyadenosine, raltitrexed, 6-mercaptopurine, hydroxyurea,6-thioguanine, decitabine (SuperGen), cytarabin, clofarabine(Bioenvision), 2-fluorodeoxy cytidine, irofulven (MGI Pharma),methotrexate, DMDC (Hoffmann-La Roche), idatrexate, or ethynylcytidine(Taiho); topoisomerase inhibitors, such as, for example, amsacrine,rubitecan (SuperGen), epirubicin, exatecan mesylate (Daiichi),etoposide, quinamed (ChemGenex), teniposide, mitoxantrone, gimatecan(Sigma-Tau), irinotecan (CPT-11), diflomotecan (Beaufour-Ipsen),7-ethyl-10-hydroxy-camptothecin, TAS-103 (Taiho), topotecan,elsamitrucin (Spectrum), dexrazoxanet (TopoTarget), J-107088 (Merck &Co), pixantrone (Novuspharma), BNP-1350 (BioNumerik), rebeccamycinanalogue (Exelixis), CKD-602 (Chong Kun Dang), BBR-3576 (Novuspharma),or KW-2170 (Kyowa Hakko); antitumor antibiotics, such as, for example,dactinomycin (actinomycin D), amonafide, doxorubicin (adriamycin),azonafide, deoxyrubicin, anthrapyrazole, valrubicin, oxantrazole,daunorubicin (daunomycin), losoxantrone, epirubicin, bleomycin, sulfate(blenoxane), therarubicin, bleomycinic acid, idarubicin, bleomycin A,rubidazone, bleomycin B, plicamycin, mitomycin C, porfiromycin,MEN-10755 (Menarini), cyanomorpholinodoxorubicin, GPX-100 (GemPharmaceuticals), or mitoxantrone (novantrone), antimitotic agents, suchas, for example, paclitaxel, SB 408075 (GlaxoSmithKline), docetaxel,E7010 (Abbott), colchicines, PG-TXL (Cell Therapeutics), vinblastine,IDN 5109 (Bayer), vincristine A, 105972 (Abbott), vinorelbine, A 204197(Abbott), vindesine, LU 223651 (BASF), dolastatin 10 (NCI), D 24851(ASTAMedica), rhizoxin (Fujisawa), ER-86526 (Eisai), mivobulin(Warner-Lambert), combretastatin A4 (BMS), cemadotin (BASF),isohomohalichondrin-B (PharmaMar), RPR 109881A (Aventis), ZD 6126(AstraZeneca), TXD 258 (Aventis), PEG-paclitaxel (Enzon,) epothilone B(Novartis), AZ10992 (Asahi), T 900607 (Tularik), IDN-5109 (Indena), T138067 (Tularik), AVLB (Prescient NeuroPharma), cryptophycin 52 (EliLilly), azaepothilone B (BMS), vinflunine (Fabre), BNP-7787(BioNumerik), auristatin PE (Teikoku Hormone), CA-4 prodrug (OXiGENE),BMS 247550 (BMS), dolastatin-10 (NIH), BMS 184476 (BMS), CA-4 (OXiGENE),BMS 188797 (BMS), or taxoprexin (Protarga); aromatase inhibitors, suchas, for example, aminoglutethimide, exemestane, letrozole, atamestane(BioMedicines), anastrazole, YM-511 (Yamanouchi), or formestane;thymidylate synthase inhibitors, such as, for example, pemetrexed (EliLilly), nolatrexed (Eximias), ZD-9331 (BTG), or CoFactor™ (BioKeys); DNAantagonists, such as, for example, trabectedin (PharmaMar), mafosfamide(Baxter International), glufosfamide (Baxter International), apaziquone(Spectrum Pharmaceuticals), albumin+³²P (Isotope Solutions), O6 benzylguanine (Paligent), thymectacin (NewBiotics), or edotreotide (Novartis);farnesyltransferase inhibitors, such as, for example, arglabin(NuOncology Labs), tipifarnib (Johnson & Johnson), lonafarnib(Schering-Plough), perillyl alcohol (DOR BioPharma), or BAY-43-9006(Bayer); Pump inhibitors, such as, for example, CBT-1 (CBA Pharma),zosuquidar trihydrochloride (Eli Lilly), tariquidar (Xenova), biricodardicitrate (Vertex), or MS-209 (Schering AG); Histone acetyltransferaseinhibitors, such as, for example, tacedinaline (Pfizer),pivaloyloxymethyl butyrate (Titan), SAHA (Aton Pharma), depsipeptide(Fujisawa), or MS-275 (Schering AG); Metalloproteinase inhibitors, suchas, for example, Neovastat (Aeterna Laboratories), CMT-3 (CollaGenex),marimastat (British Biotech), or BMS-275291 (Celltech); ribonucleosidereductase inhibitors, such as, for example, gallium maltolate (Titan),tezacitabine (Aventis), triapine (Vion), or didox (Molecules forHealth); TNF alpha agonists/antagonists, such as, for example, virulizin(Lorus Therapeutics), revimid (Celgene), CDC-394 (Celgene), entanercept(Immunex Corp.), infliximab (Centocor, Inc.), or adalimumab (AbbottLaboratories); endothelin A receptor antagonists, such as, for example,atrasentan (Abbott) YM-598 (Yamanouchi) or ZD-4054 (AstraZeneca);retinoic acid receptor agonists, such as, for example, fenretinide(Johnson & Johnson) alitretinoin (Ligand) or LGD-1550 (Ligand);immuno-modulators, such as, for example, interferon dexosome therapy(Anosys), oncophage (Antigenics), pentrix (Australian CancerTechnology), GMK (Progenics), ISF-154 (Tragen), adenocarcinoma vaccine(Biomira), cancer vaccine (Intercell), CTP-37 (AVI BioPharma), norelin(Biostar), IRX-2 (Immuno-Rx), BLP-25 (Biomira), PEP-005 (PeplinBiotech), MGV (Progenics), synchrovax vaccines (CTL Immuno),beta-alethine (Dovetail), melanoma vaccine (CTL Immuno), CLL therapy(Vasogen), or p21 RAS vaccine (GemVax); hormonal and antihormonalagents, such as, for example, estrogens, prednisone, conjugatedestrogens, methylprednisolone, ethinyl estradiol, prednisolone,chlortrianisen, aminoglutethimide, idenestrol, leuprolide,hydroxyprogesterone caproate, goserelin, medroxyprogesterone,leuporelin, testosterone, bicalutamide, testosterone propionate,fluoxymesterone, flutamide, methyltestosterone, octreotide,diethylstilbestrol, nilutamide, megestrol, mitotane, tamoxifen, P-04(Novogen), toremofine, 2-methoxyestradiol (EntreMed), dexamethasone, orarzoxifene (Eli Lilly); photodynamic agents, such as, for example,talaporfin (Light Sciences), Pd-bacteriopheophorbide (Yeda), Theralux(Theratechnologies), lutetium texaphyrin (Pharmacyclics), motexafingadolinium (Pharmacyclics), or hypericin; and tyrosine kinaseinhibitors, such as, for example, imatinib (Novartis), kahalide F(PharmaMar), leflunomide (Sugen/Pharmacia), CEP-701 (Cephalon), ZD1839(AstraZeneca), CEP-751 (Cephalon), erlotinib (Oncogene Science), MLN518(Millenium), canertinib (Pfizer), PKC412 (Novartis), squalamine(Genaera), phenoxodiol, SU5416 (Pharmacia), trastuzumab (Genentech),SU6668 (Pharmacia), C225 (ImClone), ZD4190 (AstraZeneca), rhu-Mab(Genentech), ZD6474 (AstraZeneca), MDX-H210 (Medarex), vatalanib(Novartis), 2C4 (Genentech), PKI166 (Novartis), MDX-447 (Medarex),GW2016 (GlaxoSmithKline), ABX-EGF (Abgenix), EKB-509 (Wyeth), IMC-1C11(ImClone), or EKB-569 (Wyeth).

Those additional agents may be administered separately from the acompound of formula I-containing composition, as part of a multipledosage regimen. Alternatively, those agents may be part of a singledosage form, mixed together with a compound of formula I in a singlecomposition. If administered as part of a multiple dosage regime, thetwo active agents may be submitted simultaneously, sequentially orwithin a period of time from one another normally within five hours fromone another.

The amount of both, a compound of formula I and the additionaltherapeutic agent (in those compositions which comprise an additionaltherapeutic agent as described above)) that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration.Preferably, the compositions of this invention should be formulated sothat a dosage of between 0.01-100 mg/kg body weight/day of a compound offormula I can be administered. In one example, compositions areformulated such that the dosage of a compound of formula I is from 3 to30 mg/kg body weight/day.

In those compositions that comprise an additional therapeutic agent,that additional therapeutic agent and a compound of formula I may actsynergistically. Therefore, the amount of additional therapeutic agentin such compositions will be less than that required in a monotherapyutilizing only that therapeutic agent. In such compositions a dosage ofbetween 0.01-100 mg/kg body weight/day of the additional therapeuticagent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Compounds of formula I, or pharmaceutical compositions thereof, may alsobe incorporated into compositions for coating an implantable medicaldevice, such as prostheses, artificial valves, vascular grafts, stentsand catheters. Vascular stents, for example, have been used to overcomerestenosis (re-narrowing of the vessel wall after injury). However,patients using stents or other implantable devices risk clot formationor platelet activation. These unwanted effects may be prevented ormitigated by pre-coating the device with a pharmaceutically acceptablecomposition comprising a kinase inhibitor. Suitable coatings and thegeneral preparation of coated implantable devices are described in U.S.Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.Implantable devices coated with a compound of formula I are anotherembodiment of the present invention.

Preparation of Compounds of Formula I

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

As used herein, other abbreviations, symbols and conventions areconsistent with those used in the contemporary scientific literature.See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authorsand Editors, 2nd Ed., Washington, D.C.: American Chemical Society, 1997,herein incorporated in its entirety by reference. The followingdefinitions describe terms and abbreviations used herein:

Brine a saturated solution of NaCl in water BSA bovine serum albuminDMSO dimethylsulfoxide ESMS electrospray mass spectrometry EtOAc ethylacetate EtOH ethyl alcohol HPLC high performance liquid chromatographyLCMS liquid chromatography-mass spectrometry Me methyl MeOH methanol Phphenyl RT room temperature TCA trichloroacetic acid THF tetrahydrofuranTFA trifluoacetic acid

EXAMPLE 1 2-(Quinolin-6-yl)propanoic acid (compound 1001) and2-(5,7-difluoroquinolin-6-yl)propanoic acid (compound 1002)

Compound 1001 was purchased from Okeanos Tech, Beijing, China (CatalogNo. OK-J-05025). Compound 1002 was prepared as shown in Scheme 1.Accordingly, as shown in step 1-i, to a suspension of NaH (60% inmineral oil, 8.47 g, 212 mmol) in DMSO at 0° C. (260 mL) was slowlyadded diethyl 2-methylmalonate (29.5 g, 169.4 mmol). The mixture wasstirred at 0° C. for 2 hours and 3,4,5-trifluoronitrobenzene (25.0 g,141.2 mmol) was added. The resulting mixture was warmed to RT andstirred for 12 hours. The reaction mixture was poured into saturated aq.NH₄Cl solution and the precipitate was collected by filtration. Afterwashing with water 3 times, the resulting diethyl2-(2,6-difluoro-4-nitrophenyl)-2-methylmalonate (compound 1003, 44.5 g,95% yield) was dried under reduced pressure and used as is in the nextreaction.

As shown in step 1-ii, to a solution of diethyl2-(2,6-difluoro-4-nitro-phenyl)-2-methylmalonate (44.5 g, 135 mmol) inMeOH was added Pd/C (10%, 4.0 g) under an atmosphere of nitrogen. Theatmosphere was replaced with H₂ and the mixture hydrogenated at 50 psifor 3 days. The atmosphere was replaced with nitrogen, the mixturefiltered through diatomaceous earth, and the volatiles removed underreduced pressure. The resulting diethyl2-(4-amino-2,6-difluorophenyl)-2-methylmalonate (compound 1004, 40.5 g,99% yield) was dried under reduced pressure and used as is in the nextreaction.

As shown in step 1-iii, to a solution of diethyl2-(4-amino-2,6-difluorophenyl)-2-methylmalonate (40 g, 132.8 mmol) inmethanol (200 mL) was added 6M NaOH (110.7 mL, 664.0 mmol). The mixturewas heated at 100° C. for 4 hours, cooled to 0° C., and acidified withconc. HCl until a pH of 3 was obtained. The mixture was warmed to RT andstirred for 3 hours. The resulting precipitate was collected byfiltration, washed with water, and dried under high vacuum at 50° C. for20 hours to provide 2-(4-amino-2,6-difluorophenyl)propanoic acid(compound 1005, 22 g, 84% yield): ¹H NMR (300.0 MHz, DMSO) δ 12.25 (brs,1H), 6.16 (d, J=10.8 Hz, 2H), 5.58 (s, 2H), 3.74 (q, J=7.2 Hz, 1H) and1.28 (d, J=7.2 Hz, 3H) ppm.

As shown in step 1-iv, a mixture of2-(4-amino-2,6-difluorophenyl)propanoic acid (19 g, 94.45 mmol),glycerol (35.83 g, 28.41 mL, 389.1 mmol), nitrobenzene (7.209 g, 6.028mL, 58.56 mmol) and concentrated sulfuric acid (30.57 g, 16.61 mL, 311.7mmol) was heated gently. After cessation of the initial vigorousreaction, the mixture was heated to 170° C. for 16 hours. After cooling,the volatiles were removed under reduced pressure, the residue dissolvedin MeOH (150 mL), 150 mL of 6N NaOH were added, and the mixture washeated at 110° C. for 3 hours. After cooling to RT, the mixture wasacidified with concentrated HCl to a pH of 3. The resulting darkprecipitate was collected by filtration and washed with water. Theprecipitate was taken up in ethanol and thionyl chloride (11.24 g, 6.891mL, 94.45 mmol) was carefully added dropwise. After addition wascomplete, the mixture was heated at 50° C. for 20 hours. After coolingto RT, the volatiles were removed under reduced pressure and the residuewas dissolved in a mixture of sat'd NaHCO₃ and DCM. The layers wereseparated and the aqueous layer extracted with DCM. The combinedorganics were dried over MgSO4, reduced in volume under reducedpressure, and subjected to medium-pressure silica gel chromatography (0%EtOAc/Hexanes to 30% in 36 minutes) to provide methyl2-(5,7-difluoroquinolin-6-yl)propanoate (14 g, 56% yield for two steps).The methyl ester (5.0 g) was saponified by taking it up in methanol (30mL), treating the resulting solution with NaOH (16.58 mL of 6 M, 99.50mmol), and stirring at RT for 20 hours. After careful acidification withconc. HCl to a pH of 2, the resulting precipitate was collected byfiltration and dried under high vacuum to provide2-(5,7-difluoroquinolin-6-yl)propanoic acid, which was used as is insubsequent reactions.

EXAMPLE 2 Preparation of Compounds of Formula I

The compounds of formula I are prepared as shown in Scheme 2.Accordingly, as shown in step 2-i of Scheme 2, the appropriatelysubstituted quinoline acetic acid of formula II (248.5 mmol, 1.0equivalent) and 1,3-diaminothiourea (273.4 mmol, 1.1 equivalents) aresuspended in a mixture of tetramethylene sulfone (sulfolane, 38 mL) andwater (57 mL). Methane sulfonic acid (546.7 mmol, 2.2 equivalents) isadded to the mixture, whereupon all solids dissolve. The reactionmixture is slowly warmed to 90° C. and the reaction heated at 90° C. for40 hours, monitoring the progress of the reaction HPLC analysis. Thereaction mixture is cooled in an ice bath and water (75 mL) is added,followed by the careful addition of saturated sodium bicarbonate (500mL) until a pH 8 is achieved. The resulting precipitate is collected byvacuum filtration, washed with water, saturated sodium bicarbonate,water, and methyl t-butyl ether, respectively. The product is dried in avacuum oven at 55° C. to afford a compound of formula III.

As shown in step 2-ii of Scheme 2, a compound of formula III (453.3mmol, 1.00 equivalent) and 1-methyl-1H-pyrazole-4-carboxylic acid (476.0mmol, 1.05 equivalents) are dissolved in POCl₃ (1.23 liters) andsulfolane (246 mL). The resulting mixture is stirred for 18 hours at 83°C. The volatiles are evaporated under reduced pressure and the residueadditionally azeotroped with toluene twice more under reduced pressure.The resulting oil is slowly poured into stirring ice-water and theaqueous solution is extracted with dichloromethane to remove anyresidual sulfolane. The aqueous solution is treated with saturatedsodium bicarbonate (3.2 liters) until a pH of 7 is achieved. Theresulting oil is decanted off and dissolved in a small amount ofmethanol, with the remaining aqueous layer extracted withdichloromethane (4 times). The combined organic extracts and themethanol solution of the oil are combined and washed with saturatedsodium bicarbonate, water, and brine, respectively. The organics aredried over anhydrous MgSO₄, filtered, and evaporated under reducedpressure to afford the crude product as a thick brown oil. The productis purified by silica gel chromatography, eluting with a gradient ofdichloromethane to 5% methanol in dichloromethane. Fractions containingproduct are evaporated under reduced pressure to provide compounds offormula Ia, which can be further purified by crystallization fromdichloromethane and methyl t-butyl ether. The racemic mixtures ofcompounds of formula Ia were taken up in methanol (33 mg/mL) containing1% TFA and separated into their respective enantiomers by supercriticalfluid chromatography (SFC). Accordingly, compound 1 and its enantiomerwere separated using a ChiralPak® AD-H column (80 μL injections; 20mm×250 mm, 5 micron column; eluted with 25% MeOH (0.1% DEA), 75% CO₂; 70mL/min at 100 bar). Under these conditions, the (S)-enantiomer (compound1a) eluted first and the (R)-enantiomer (compound 1) eluted second.Compound 2 and its enantiomer were separated using a ChiralCel® OJ-Hcolumn (100 μL, injections; 20 mm×250 mm, 5 micron column; eluted with10% MeOH (0.1% DEA), 90% CO₂; 60 mL/min). Under these conditions, the(S)-enantiomer (compound 2a) eluted first and the (R)-enantiomer(compound 2) eluted second. Analytical data for compounds of theinvention are shown in Table 1.

TABLE 1 Physical Characterization of Compounds of Formula I ¹H NMR (300MHz, unless Cmpd. Rotation ESMS indicated otherwise), NMR No. [α]_(D) ²³(M + H) peaks given as δ values 1 +161 362.07 (DMSO-d₆) δ 9.17-9.15 (m,1H), (0.610 g/100 mL 8.94 (d, J = 8.1 Hz, 1H), 8.55 MeOH) (s, 1H), 8.23(d, J = 8.9 Hz, 1H), 8.22 (s, 1H), 8.10 (d, J = 8.7 Hz, 1H), 8.04 (d, J= 0.6 Hz, 1H), 7.92 (dd, J = 5.0, 8.2 Hz, 1H), 4.99 (q, J = 7.3 Hz, 1H),3.90 (s, 3H) and 1.88 (d, J = 7.2 Hz, 3H) ppm 2 +145 398.24 (DMSO-d₆) δ9.01 (dd, J = 1.5, (0.985 g/100 mL 4.3 Hz, 1H), 8.53 (d, J = 7.8 Hz,MeOH) 1H), 8.45 (s, 1H), 7.87 (s, 1H), 7.75 (d, J = 11.7 Hz, 1H), 7.65(dd, J = 4.3, 8.6 Hz, 1H), 5.18 (q, J = 7.1 Hz, 1H) and 1.95 (d, J = 7.2Hz, 3H) ppmBiological Assay of Compounds of Formula I

EXAMPLE 3 c-Met Kinase Inhibition Assay

The compounds of the invention were screened for their ability toinhibit c-Met kinase using a standard radiometric assay. Briefly, inthis kinase assay the transfer of the terminal ³³P-phosphate in ³³P-ATPto substrate polyE4Y is interrogated. The assay was carried out in96-well plates to a final volume of 100 μL per well containing 0.5 nMc-Met, 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 0.01% BSA, 1 mMDTT, 0.5 mg/mL polyE4Y, and 35 μM ATP. Accordingly, compounds of theinvention were dissolved in DMSO to make 10 mM initial stock solutions.Serial dilutions in DMSO were then made to obtain the final solutionsfor the assay. A 1.5 μL aliquot of DMSO or inhibitor in DMSO was addedto each well, followed by the addition of ³³P-ATP, and finally theaddition of c-Met and polyE4Y (obtained from Sigma). After 20 min, thereaction was quenched with 50 μL of 30% trichloroacetic acid (TCA)containing 4 mM ATP. The reaction mixture was transferred to the 0.66 mmGF filter plates (Corning) and washed three times with 5% TCA. Followingthe addition of 50 μL of Ultimate Gold™ high efficiency scintillant(Packard Bioscience), the samples were counted in a Packard TopCount NXTMicroplate Scintillation and Luminescence Counter (Packard BioScience).The K_(i) values, shown in Table 2, were calculated using MicrosoftExcel Solver macros to fit the data to the kinetic model for competitivetight-binding inhibition.

EXAMPLE 4 Inhibition c-Met Activity in Snu5 Gastric Carcinoma Cells

Compounds of formula I were also screened for their ability to inhibitthe Luciferase-induced signal in an engineered Snu5 cell line. Snu5[obtained from American Type Culture Collection (Catalog numberCRL-5973)] is a human gastric carcinoma known to overexpress c-Met,which is constitutively active. The cell line was transduced with theretrovirus, pCLPCX, which contains a genetic construct consisting of6×AP 1 promoter response elements and a luciferase gene having aC-terminal PEST sequence (proteolytic signal from mouse ornithinedecarboxylase, which reduces the half-life of the luciferase). Theconstitutively active c-Met activates cellular pathways (principally MAPkinase), resulting in AP-1-induced transcription of luciferase-PEST andtranslation into the final product, the activity of which isquantifiable as a chemiluminescent readout upon the addition ofluciferin (Steady-Glo from Promega). Residual luminescence is stronglycorrelated to the inhibition of c-Met. A stable cell line was obtainedby selecting the new cell line (Snu5-AP1-Luc-Pest) with puromycin. Thecells were grown in complete media [Iscove's media (Invitrogen)containing 10% fetal bovine serum (FBS, Hyclone) andpenicillin/gentamycin (Invitrogen)]. Compounds of the invention weredissolved in DMSO to make 10 mM initial stock solutions. Serialdilutions in DMSO were then made and transferred to complete medium tomake a 10× solution. The Snu5-AP1-Luc-Pest cells were counted anddiluted to 200,000-cells/mL solution. The cells (90 μL) were added toeach well in a 96-well black with clear bottom plate (Costar). Then 10μL of the 10× compound solution was added to the cells in triplicate.The plates were incubated in a 37° C./5% CO₂ incubator. After 6 hours,50 μL of the Steady-Glo reagent (Promega) was added to each well andplaced on a plate shaker for 5 minutes to ensure that the cells werecompletely lysed. The plate was read on a 1450 Microbeta LiquidScintillation and Luminescence Counter (Perkin-Elmer). The IC₅₀ values,shown in Table 2, were calculated using a 4-parameter fit using thegraphing software Prism (GraphPad).

EXAMPLE 5 Inhibition of PDE3

Phosphodiesterase-3 (PDE3) is one of a family of phosphodiesterasesresponsible for the regulation of cyclic nucleotide second messengers.PDE3 enzymes are involved in regulation of cardiac and vascular smoothmuscle contractility. Agents that inhibit PDE3 were originallyinvestigated for the treatment of heart failure but have unwantedarrhythmic side effects. See Dart R. C., Medical Toxicology, Edition 3,page 708; Lippincott 2004.

Compounds of formula I were screened for their ability to inhibit PDE3in a mobility-shift assay that quantitates the conversion of aflourescent analog of cyclic AMP to a flourescent analog of AMP using aLabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.). Accordingly, a0.6 μL aliquot of the test compound dissolved in DMSO was placed in awell of a 384-well plate, followed by the addition of a 15 μL aliquot of200 ng/mL PDE3A (BPS Bioscience, San Diego, Calif., Catalog No. 60030)in 50 mM HEPES buffer at pH 7.5, containing 5 mM MgCl₂ and 0.001%Brij-35 (Buffer A). The enzyme and compound were incubated at roomtemperature for 15 minutes, followed by the addition of a 15 μL aliquotof 2 μM iFL-cAMP (Caliper Life Sciences, Catalog No. 760444) in BufferA. Following a 40 minute incubation at room temperature, the reactionwas quenched by the addition of a 45 μL aliquot of 100 mM HEPES bufferat pH 7.5, containing 30 mM EDTA, 0.015% Brij-35, and 0.1% CR-3 (CaliperLife Sciences). Separation of substrate and product was achieved in arunning buffer consisting of ProfilerPro Separation Buffer (Caliper LifeSciences, Catalog No. 760367), 50 μM CR-8 (Caliper Life Sciences,Catalog No. 760278), and 0.1% CR-3 using a 12-sipper chip on a LabChip3000 with a downstream voltage of −2000 volts, an upstream voltage of−250 volts, and a running pressure of −2.0 psi. Percent conversion wasdetermined using HTS Well Analyzer software (Caliper Life Sciences, v5,SP2). IC₅₀ values were determined using non-linear regression with Prism(v5.0a, GraphPad Software, Inc., La Jolla, Calif.). The IC₅₀ values ofcompounds 1 and 2, along with their respective enantiomers 1a and 2a,are shown in Table 2

TABLE 2 SNU5- c-Met AP1 PDE3 Cmpd. No. Ki (nM) IC₅₀ (nM) IC₅₀ (nM)Structure 1  <300 <100 6600

1a <300 <100  760

2  <300 <100 6150

2a <300 <100  150

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity orunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein each R¹ is,independently, hydrogen or fluoro.
 2. The compound according to claim 1,wherein R¹ is hydrogen.
 3. The compound according to claim 1, wherein R¹is fluoro.
 4. A pharmaceutical composition comprising a compoundaccording to any one of claims 1 to 3, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier, adjuvant orvehicle.
 5. The composition according to claim 4, additionallycomprising a chemotherapeutic or anti-proliferative agent, ananti-inflammatory agent, an agent for treating atherosclerosis, or anagent for treating lung fibrosis.
 6. A method of treating or lesseningthe severity of metastatic cancer in a patient comprising administeringthe compound according to claim 1, or a pharmaceutical compositioncomprising said compound, in an amount sufficient to treat or lessen theseverity of said metastatic cancer in said patient.
 7. A method oftreating or lessening the severity of hepatocellular carcinoma in apatient comprising administering the compound according to claim 1, or apharmaceutical composition comprising said compound, in an amountsufficient to treat or lessen the severity of said hepatocellularcarcinoma in said patient.